A central problem in metazoan development is how cells are specified to develop along one particular pathway. I propose to analyze two developmental controls in the nematode, Caenorhabditis elegans: specification of cell type (sperm or oocyte) and regulation of cell proliferation (choice between mitosis and meiosis in the germ line). During the first period of this grant, my lab identified genes central to both controls. In addition, we proposed a model for the genetic control of germ-line sex determination that provides an intellectual framework for many of the experiments presented here. In the next five years, I plan an intensive genetic analysis of sex determination in the germ-line tissue of this nematode. Most, and perhaps all, genes central to the sperm/oocyte decision will be identified, and a genetic and phenotypic characterization of mutants will be used to study the function of the sex determination control genes. In addition, a molecular analysis of sex determination control will be initiated by cloning the fem-3 gene. This locus was selected because the properties of fem-3 mutants suggest that it is central to sex determination in all tissues. Once cloned, fem-3 gene structure and expression will be examined in wild-type and mutant animals. A genetic analysis of the germ-line choice between continued mitoses and entry into meiosis is also planned. Genes central to this decision will be identified by mutation, and the function of each gene will be studied by genetic and phenotypic characterization of mutant strains. For analysis of gene function in individual tissues during development several techniques will be used. Microinjection and in situ hybridization will be employed to introduce and localize fem-3 transcripts to explore the cellular basis of that control. Blastomere injections and genetic mosaics will be used to study the tissue specificity of genes not yet clone.d The health relatedness of this work derives from its contribution to an understanding of mechanisms of control over cell growth and differentiation. Defects in such controls may lead to congenital defects or cancer in humans.